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Abstract

Background

Thick blood films are routinely used to diagnose Plasmodium falciparum malaria. Here, they were used to diagnose volunteers exposed to experimental malaria
challenge.

Methods

The frequency with which blood films were positive at given parasite densities measured
by PCR were analysed. The poisson distribution was used to calculate the theoretical
likelihood of diagnosis. Further in vitro studies used serial dilutions to prepare thick films from malaria cultures at known
parasitaemia.

Results

Even in expert hands, thick blood films were considerably less sensitive than might
have been expected from the parasite numbers measured by quantitative PCR. In vitro work showed that thick films prepared from malaria cultures at known parasitaemia
consistently underestimated parasite densities.

Conclusion

It appears large numbers of parasites are lost during staining. This limits their
sensitivity, and leads to erroneous estimates of parasite density.

Background

Microscopy of thick blood films is the usual diagnostic test for Plasmodium falciparum malaria. Density is usually assessed by thick films, either by counting parasites
per microscope field, or by counting parasites per hundred white blood cells [1]. Thick films contain several layers of red cells, whereas thin films contain a single
layer of spread red cells. Thus, for a fixed number of microscope fields, thick films
allow the microscopist to examine a larger number of red cells for the presence of
parasites, and low parasitaemias can be more readily identified by thick film. Thin
films are preferred to examine the morphology of parasites and determine species.
Non-immune individuals may be unwell when one parasite or less is present in an entire
thick film, requiring laborious, repeated examinations to make a diagnosis.

Sporozoite challenge experiments were conducted, where volunteers were exposed to
experimental malaria challenge to assess candidate malaria vaccines [2-5]. Treatment decisions were based on blood film analysis, but PCR was conducted on
all blood samples [6]. PCR data was not made available until after the trial was been completed.

The sensitivity of thick blood films was studied using data obtained during these
trials, compared this with quantitative PCR data, and further investigated these findings
with in vitro studies.

Methods

Sporozoite challenge

Volunteers gave informed consent. Procedures were reviewed by OXREC (Oxford Research
Ethics Committee), the local ethics committee, and were in accordance with the declaration
of Helsinki (revised 1983). Twice daily blood samples were taken from day 6 until
day 14, then daily until day 21. At least 100 high powered fields of a thick blood
film were viewed and quantitative PCR performed on each sample. Volunteers were treated
when a single parasite was seen by blood film, after the appearance of the parasite
was confirmed by a second microscopist. Neither managing clinicians nor microscopists
were aware of PCR data during the trial.

Thick blood films

Giemsa staining was used for the first two sporozoite challenge studies, and Field's
stain in coplin jars for the later two studies. The thick film was air dried in both
methods. For giemsa staining, the film was stood in 5% Giemsa for 30 minutes, then
washed gently in tap water and air dried. Field's stain was applied by dipping the
slide into Field's stain A for 3 seconds, then into tap water for 3 seconds (with
gentle agitation), into Field's stain B for a further 3 seconds and then washing gently
in tap water to remove excess stain. The slide was then air dried for at least 30
minutes. The lead microscopist held a post in the London School for Hygiene and Tropical
Medicine Clinical Parasitology Laboratory, the UK national reference laboratory, and
others at the Medical Research Council, the Gambia. The lead microscopist examined
slides produced by serial dilutions, blind to source. The average thick film uses
10 μl of blood spread over one thousand high powered fields, so the 100 high powered
fields routinely examined during views 1 μl of blood [7].

PCR

The PCR method is described elsewhere [6]. Briefly, EDTA anticoagulated blood samples were filtered to remove leukocytes, DNA
was purified from 0.5 ml filtered blood, and eluted into 50 μl. A portion of the multicopy
18S (small subunit) ribosomal RNA genes of P. falciparum was amplified by PCR and the increase in PCR product detected by binding the fluorescent
dye SYBR Green I using the Rotor-Gene Real-Time PCR machine (Corbett Research), using
l μl extracted DNA in duplicate. The increase in PCR product is quantitated by comparison
with standard preparations of known parasite numbers.

Results

Sensitivity of blood films during sporozoite challenge

The poisson distribution was used to calculate the likelihood of sampling a parasite
within the blood volume examined in microscopy, at given parasite densities identified
by PCR. At low parasitaemias there was a discrepancy between the likelihood of diagnosis
calculated by PCR readings, and the actual frequency of diagnosis at that density
by thick film. For the thick films prepared between 100 and 1000 parasites per ml,
the overall calculated likelihood of sampling a parasite in 1 μl was 26%. However,
thick films had a sensitivity of only 9% (95% CI 4–15%) in this range. Between 1,000
and 10,000 parasites per ml, the calculated likelihood of a parasite being present
was 84%, but the actual sensitivity of thick films was 29% (CI 19–39%). It was only
above 10,000 parasites per ml when thick films had higher sensitivity (81% CI 65–97%),
when the calculated probability of sampling was 99%.

This surprising finding suggested that a significant number of parasites were not
visualized on a thick film despite being theoretically present in the original blood
sample used to make the film. Results did not vary according to staining protocol
(Giemsa or Field stain) or by microscipist. A similar density threshold for reliable
diagnosis of malaria by thick film examination is reported elsewhere [8].

Serial dilution

Experiments using serial dilution of a known parasite density were then conducted
to extend this observation. An in vitro culture of Plasmodium falciparum was prepared at 5–10% parasitaemia. The parasite count was first accurately determined
by a thin film (in duplicate). The culture was then serially diluted with uninfected,
fresh whole blood. Red cell counts were made by Coulter™ counter for both the original
culture and the uninfected blood used for serial dilutions. This allowed accurate
calculation of predicted parasite numbers, without having to count thin films at each
dilution. At each serial dilution, PCR analysis was conducted on 0.5 mls blood, and
2 thick films made, using exactly 10 μl. The whole film was read, blind to source.

Densities seen by blood film during serial dilution

For the serially diluted parasite cultures, the parasite density measured by PCR and
thick film was compared with that calculated from serial dilution (Fig 1). Although PCR readings corresponded well with actual parasite numbers generated
from serial dilutions, thick films were less reproducible, but tended to measure parasite
densities approximately one log lower than those calculated by serial dilution.

Figure 1. At each serial dilution (x axis), parasite densities seen by PCR (open circles) and
parasite densities seen by thick blood film examination (filled circles) are both
plotted on the y axis. The PCR readings are the result of a single experiment, the
thick film readings are the results of two experiments. The solid line (least squares
regression line for PCR results against densities known from serial dilution) is given
by y = 0.98x + 0.07 (95% CI -0.12 to 0.27). The dotted line (regression line for thick
film densities against serial dilution, ignoring the outlier) is given by y = 0.78x
- 1.5 (95% CI -0.61 to -2.4) (y = 0.81x - 1.26, 95% CI - 0.07 to -2.5 including the
outlier). Densities measured by thick film are therefore approximately 1 log lower
than those calculated by serial dilution, whereas PCR readings match the serial dilution
more closely.

Discussion

It is unlikely that cells or parasites are hidden during microscopy, since adequate
preparation of the slide ensures visibility through all planes of focus. It is more
likely that parasites are either washed off or lysed during staining, since PCR of
the staining reagents to detect parasites was positive, and transfer of parasites
from positive slides to negative slides during staining has been recognized for some
time [8].

Thin film and thick film parasite density estimates have been compared in previous
studies. Although thick films are more sensitive than thin films, they significantly
underestimated the parasite density in some studies [7,9,10], but not in others [11]. These previous studies lacked accuracy, since at high parasitaemias thick films
are difficult to count accurately, and thin films cannot be counted accurately at
low parasitaemias. In the study presented here, this difficulty was avoided by using
serial dilution to provide known concentrations of parasites, and the accuracy of
serial dilution was confirmed by quantitative real time PCR. PCR counts gene copy
number, and this might have led to an over-estimate of parasite numbers when counting
multi-nucleated schizonts. However, only mononucleated parasites are identified in
peripheral blood at the low parasitaemias seen in this study, and in vitro cultures were synchronous.

Conclusion

Thick films are considerably less sensitive than might be possible and underestimate
parasite densities. In routine clinical work in non-endemic areas the loss in sensitivity
makes thick film examination much more laborious, and a fixation method that prevented
parasites being washed off would improve the sensitivity of the method. In malaria
endemic areas, the loss in sensitivity is not critical, since semi-immune patients
are likely to have a high parasitaemia if they present with febrile malaria. In epidemiological
studies, parasite density thresholds are defined to distinguish febrile malaria from
chronic parasitaemia [12,13]. A systematic underestimate of densities would not alter the classification of individuals,
but if some density counts are made by thick and some by thin film, and this will
lead to an underestimate of the lower parasitaemias counted by thick film. Furthermore,
should epidemiological studies to define parasite densities be repeated by quantitative
PCR, considerably higher parasite density thresholds will be identified to define
malaria cases. These will not be comparable to previous studies.

Authors' contributions

Experimental Design: PB, FS, SG

Experimental work: LA, FS, AHC.

Writing of Manuscript PB, SG, AVSH.

Acknowledgements

The study was performed with the permission of OXTREC, the Oxford University Tropical
Research Ethics Committee, and funded by the Wellcome Trust. P Bejon holds a Wellcome
Trust training fellowship (073597) and AVSH is a Wellcome Trust Principal Research
Fellow. There are no conflicts of interest. F Sanderson has since moved to Imperial
College School of Medicine.